Selenium is a trace element whose biological activity is manifested in the form of the 21st amino acid selenocysteine (Sec), and its incorporation into an essential class of proteins called selenoproteins. Many selenoproteins are antioxidants that are intimately involved in the protection of DNA, lipids and proteins from oxidative damage. Oxidative damage arising from reactive oxygen species has been implicated in aging and a number of human diseases including cancer and cardiovascular disease. The ultimate goal of our research is to completely elucidate the mechanism for selenocysteine incorporation so that we could in turn manipulate the levels of these beneficial selenoproteins. Sec incorporation represents a unique process because unlike other amino acids, it is encoded by a UGA codon which canonically signals for translation termination. Several cis and trans-acting factors are essential for the "receding" of UGA; the known factors include a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region of all selenoprotein mRNAs, a SECIS binding protein (SBP2), and a Sec-specific elongation factor (eEFSec) that binds and delivers the Sec-tRNA[Ser]Sec. While many of the factors have been identified, the precise mechanism and the order of events have not been determined. In addition to binding the SECIS element, SBP2 has also been reported to bind stably and quantitatively to ribosomes in vivo. Our research focuses on SBP2 and its communication with the ribosome. Specifically, this proposal seeks to determine the significance of the SBP2-ribosome interaction and to characterize the SBP2 binding site on the ribosome. We will make use of an in vitro ribosome binding assay to test SBP2 mutants for defects in ribosome binding. The functional significance of these mutations will be investigated by assessing the effect on selenoprotein expression in cells and on Sec incorporation in a luciferase reporter construct in vitro. To characterize the binding site of SBP2 on the ribosome, mammalian rRNA fragments will be generated and tested for their ability to bind SBP2 by gel shift analysis and competition studies. The results obtained will aid in piecing together the mechanism for Sec incorporation and thus provide therapeutic targets to allow for the careful manipulation of selenoprotein expression in the treatment and prevention of disease. [unreadable] [unreadable] [unreadable]